1. Field of the Invention
This invention relates to a recombinant plasmid (a hybrid plasmid) permitting efficient expression of L-phenylalanine ammonia-lyase (hereinafter abbreviated as PAL) in Escherichia coli, and a strain of E. coli which has been transformed with the recombinant plasmid.
More particularly, the invention relates to a hybrid plasmid characterized in that, when the hybrid plasmid is constructed by inserting the structural gene for PAL in an expression vector enabling the expression of an exogenous gene in E. coli according to gene manipulation techniques, a combined promoter obtained by connecting, in a specified order, (a) a hybrid promoter (the tac promoter) constructed from the trp promoter minus 35 region and the lac UV-5 promoter minus 10 region with (b) the P.sub.L promoter of the lambda phage, the so constructed tandem promoter is used as the promoter for the expression of the structural gene for PAL. By using the hybrid plasmid, more efficient expression of PAL in E. coli can be achieved. This invention further relates to a strain of E. coli which has been transformed with the hybrid plasmid.
2. DESCRIPTION OF THE PRIOR ART
PAL is an enzyme catalyzing the reaction in which ammonia is removed from L-phenylalanine to form trans-cinnamic acid. Accordingly, it is useful in the production of L-phenylalanine from cinnamic acid and ammonia by utilizing the reverse reaction.
In the past, PAL has been prepared by extraction from yeasts, molds and plants. However, such organisms that produce PAL can only give a slight yield of PAL, and it has been difficult to prepare PAL on an industrial scale.
Accordingly, as a means for realizing the mass production of PAL, much attention is focused on the genetic recombination technique which enables mass-culturable microorganisms (such as E. coli and the like) to produce PAL.
From this point of view, the present inventors have elucidated the construction of the structural gene for PAL derived from Rhodosporidium toruloides and have succeeded in causing PAL (i.e., the product of the structural gene) to be expressed in E. coli.
Meanwhile, as a host microorganism serving to produce an exogenous protein (i.e., a protein which is not normally produced by the host microorganism) by utilization of the genetic recombination technique, E. coli is being widely used because its biological properties have been fully analyzed, it is not pathogenic, and it can be readily cultured in a medium having a simple composition.
As an expression vector allowing the desired protein to be expressed in E. coli, the vector used is one which basically comprises a promoter permitting the transcription, in E. coli, of a DNA sequence located downstream of the promoter and containing the region coding for the desired exogenous protein, and a vector capable of replicating in E. coli.
Various types of promoters are available for use as the promoter incorporated in such an expression vector. For example, the P.sub.L promoter of the lambda phage of E. coli (hereinafter referred to as the P.sub.L lambda promoter), the promoter of the tryptophan operon of E. coli (hereinafter referred to as the trp promoter), the promoter of the lactose operon of E. coli (hereinafter referred to as the lac promoter), and the fusion promoter (hereinafter referred to as the tac promoter) composed of the trp promoter and the lac promoter are being widely used for that purpose.
Moreover, in order to produce exogenous proteins more efficiently by the use of expression vectors, further investigations are required to search for a new, highly active promoter permitting more efficient expression and/or enhance the activity of conventionally known promoters.
For example, K. Mackenney et al. made an attempt to obtain a more active promoter by connecting a plurality of promoters in series [Gene Amplification and Analysis, Vol. II, pp. 383-415, Elsevier Science Publishing Co., New York (1981)]. Moreover, in order to accomplish the same purpose, Japanese Patent Laid-Open No. 126086/'85 discloses tandem promoters constructed by connecting the P.sub.L lambda promoter or the P.sub.R promoter (i.e., the P.sub.R promoter of the lambda phage of E. coli) in series with the trp promoter or the lac promoter so that the former is located upstream of the latter.
However, the compatibility of the promoter incorporated in an expression vector with the protein to be expressed thereby has not been fully elucidated yet. Accordingly, it is very difficult to make a theoretical presumption, for example, as to whether or not a promoter having high activity for the expression of some proteins can also exhibit high activity for the expression of other proteins. Moreover, much remains unknown about the action of a tandem promoter used to enhance expression efficiency. Thus, the types of promoters to be connected and the order of connection of them must be determined according to the type of a protein to be expressed.
Accordingly, it is necessary to select or develop a promoter permitting more efficient expression, for each of the proteins to be expressed.
For these reasons, the selection or development of a more suitable promoter for the expression of PAL in E. coli has been urgently needed.
Moreover, it has been found that the conventionally known promoters, such as those enumerated above, fail to provide satisfactorily high expression efficiency. Thus, there is a need to develop a new promoter suitable for high-level expression of PAL.